As a method for quantitatively determining homocysteine in a specimen, a method wherein the homocysteine is reacted with an enzyme and the formed hydrogen sulfide is measured, has been known. The enzyme used in this method is, for example, L-methionine-γ-lyase or homocysteine desulfhydrase. Further, the present inventors have found that o-acetylhomoserine-lyase (EC class 4.2.99), known as an enzyme involved in the amino acid biosynthesis, also can produce hydrogen sulfide by catalyzing γ-substitution reaction in the presence of a thiol compound, and reported that homocysteine can be quantitatively determined with this enzyme (see JP-A-2000-166597).
However, since these enzymes react not only with homocysteine but also with cysteine to form hydrogen sulfide, there is a problem that it is difficult to measure accurate homocysteine concentration in a specimen wherein homocysteine and cysteine coexist (for example, a biological specimen such as blood).
Then, the present inventors proposed a method wherein, while the relation between known cysteine content of a starting solution and the amount of hydrogen sulfide resulting from a reaction with the above enzyme is preliminarily formulated, a specimen containing cysteine and homocysteine is reacted with said enzyme under a predetermined condition to measure the amount of formed hydrogen sulfide (1), and then the amount of hydrogen sulfide (2) that is derived from cysteine contained in said biological specimen and estimated, by calculation with the above formula, from a separately determined cysteine content in said biological specimen, is subtracted from the amount of hydrogen sulfide in (1) to determine the net content of homocysteine in the biological specimen wherein cysteine coexists (JP-A-2000-270895).
Further, Japanese International Publication No. 2000-513589 discloses a method wherein cysteine in a biological specimen is pretreated by an enzyme (e.g. γ-glutamyl cysteine synthase, cysteine oxidase, cystathionine β synthase or cysteine tRNA synthase) which is capable of converting it to a compound which is not a substrate of homocysteinase, and wherein the remaining homocysteine is measured with homocysteinase.
However, the method as disclosed in JP-A-2000-270895 is not a simple method since the cysteine content must be determined separately.
On the other hand, the method as disclosed in Japanese International Publication No. 2000-513589 has the following problems.
For example, the γ-glutamyl cysteine synthase (derived from rat kidney) has low substrate specificity, and may react not only with cysteine but also with cysteine derivatives or DL-homocysteine (see Biochemistry, 10(3), 1971; 372–380).
Further, although the cystathionine β synthase is known to be capable of undergoing a β-substitution reaction toward cysteine in the presence of a thiol compound, it can not be used for a method wherein homocysteine concentration is estimated by measuring the amount of formed hydrogen sulfide resulting from a reaction of homocysteine with an enzyme, because it produces hydrogen sulfide as product.
Moreover, since the cysteine tRNA synthase uses RNA as the substrate, there is a problem that it is susceptible to RNase.
Furthermore, cysteine dioxygenase derived from rat liver (another name: cysteine oxidase) has problems, for example, (1) it is necessary to have the enzyme incubated in the presence of cysteine under an anaerobic condition in order to convert it to an active form, (2) since it is very unstable and tends to be inactivated, its storage is difficult without a stabilizing protein (Protein-A) (see J. Biochem. 83(2), 1978; 479–491), and (3) it is easily inactivated in the presence of a reducing agent.
Accordingly, it is difficult to eliminate the cysteine in the specimen simply and efficiently by the methods disclosed in the above-mentioned patent publications.
Further, Japanese International Publication No. 2000-513589 discloses methionine γ-lyase derived from Trichomonas vaginalis, of which the substrate specificity toward homocysteine is enhanced by introducing mutation. JP-A-2002-10787 discloses o-acetylhomoserine sulfhydrase which is one of the enzymes involved in the amino acid biosynthesis derived from Thermus thermophilus, as an enzyme which does not substantially react with cysteine and specifically reacts with homocysteine. However, since there is a case where the cysteine concentration in the biological specimen is considerably high exceeding 1 mM, the use of such enzyme having improved substrate specificity is not sufficient to avoid the influence of cysteine completely.